Approximately 18 million Americans have diabetes resulting in an estimated $92 billion in direct medical costs per year. Type 2 diabetes mellitus accounts for approximately 90-95% of all diabetes. Although the cause of type 2 diabetes is unknown, one common factor of the disease is insulin resistance. Since skeletal muscle is the primary site for insulin-mediated glucose disposal, insulin resistance is thought to be due, in part, to a defect in skeletal muscle's ability to respond appropriately to increases in circulating insulin. The goal of this proposal is to determine whether or not the mammalian target of rapamycin (mTOR) plays a role in the development of skeletal muscle insulin resistance. mTOR is a nutrient sensitive and insulin responsive serine/threonine kinase that controls mRNA translation and cellular growth. Recent cell culture studies have demonstrated that activating the mTOR pathway inhibits insulin signal transduction and promotes insulin resistance. Our first hypothesis is that activating mTOR-mediated signal transduction will produce insulin resistance in skeletal muscle, a process that will be reversed by rapamycin, a highly specific inhibitor of mTOR. Specific aim #1 will examine the effects of mTOR-mediated signaling on insulin action in skeletal muscle. We will determine if incubating muscle with amino acids produces insulin resistance by an mTOR dependent serine phosphorylation of the insulin receptor substrate-1 (IRS-1). Serine phosphorylation of IRS-1 is thought to promote insulin resistance by decreasing IRS-1 tyrosine phoshorylation and promoting IRS-1 degradation. We will also examine the effects of activating mTOR with amino acids on insulin's ability to promote the phosphorylation of protein kinase B (PKB) on Thr308 and Ser473. Phosphorylation of PKB on these sites is necessary for full kinase activity and the stimulation of glucose transport. Our preliminary data indicate activating mTOR signaling reduces insulin-stimulated PKB phosphorylation on Ser473 in skeletal muscle, a process that is abolished by rapamycin. Finally, specific aim #1 will determine if activating mTOR signaling with amino acids decreases insulin's ability to recruit GLUT4 to the cell surface and stimulate glucose transport in skeletal muscle. Our second hypothesis is that the hyperinsulinemia associated with diet-induced insulin resistance constitutively activates mTOR. Specific aim #2 will examine the phosphorylation of mTOR on Ser2448 in muscles from mice subjected to a high fat diet compared to controls. Ser2448 is a site in mTOR whose phosphorylation is associated with increased kinase activity. The results from these studies will provide new insights into factors that play a role in the development of insulin resistance and possibly lead to the use of mTOR inhibitors as novel treatments for type 2 diabetes. [unreadable] [unreadable]